Comparison of biofilm dispersal approaches in Pseudomonas aeruginosa and evaluation of dispersed cells in acute infection mouse model

Abstract

Biofilm dispersal is biologically significant process to fully understand its consequences during biofilm based infections. The mechanism for biofilm dispersal may involve response to environmental cues and changes in intracellular secondary messengers. Considering range of cues for biofilm dispersal, it is significant to study if dispersed cells generated by different methods have similar phenotype. In the present study, we have compared four type of biofilm dispersal in P. aeruginosa based in response to environment cues (starvation and nutrient rich), c-di-GMP knockdown and signaling molecule NO. We have determined their dispersal efficiency, susceptibility of dispersed cells for antibiotics, their transcriptomic changes compared to planktonic and biofilm. We also determined if dispersed cells can cause acute infections in mouse models. In vitro experiments showed that NO and c-di-GMP based dispersal methods had high biofilm reduction efficiency of 50 and 75% respectively, however, the nutrient induced dispersion showed low efficiency (≈30%) and more tolerance to colistin.We also showed that in vitro dispersed cells induced >10-fold transcriptomic expression of genes (at significance level of p < 0.005) related to efflux pumps (mexCD-oprJ), antibiotic resistance (arnDET) in dispersed cells induced by NO and nutrient change, while denitrification pathway and virulence (T3SS, VreI, T2SS) genes in all dispersed cells compared to planktonic and biofilm state. When the ability of the dispersed cells was tested in mouse model of lung infection, c-di-GMP and NO based dispersed cells displayed enhanced infections and haematogenous spread to liver and spleen and higher mortality. Although degree of immune response (cytokines) does not differ based on phenotypes inoculated in our experimental conditions. Based on our data most efficient dispersal methods increase murine mortality. This indicates their capability of making biofilm associated infection more complicated. Our data encourage to be more careful for studies suggesting biofilm dispersal during treatment of biofilm infections.